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Boon Chin Heng

Publications and source records attributed to Boon Chin Heng.

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Feeder cell density--a key parameter in human embryonic stem cell culture.

A key issue in human embryonic stem (ES) cell culture that has largely been ignored is the high degree of variability in the murine embryonic fibroblast (MEF) feeder cell density, which has been reported by different studies and protocols. Presumably, too low a feeder cell density would result in insufficient levels of secreted factors, extracellular matrix, and cellular contacts provided by the feeder cells for the maintenance of human ES cells in the undifferentiated state. Too high a feeder cell density, on the other hand, may result in a more rapid depletion of nutrients and oxygen within the in vitro culture milieu, as well as physically hinder the attachment and growth of ES colonies during serial passaging. Preliminary investigations by our group revealed that an elevated MEF cell density of 32,000 cells/cm2, above the recommended value of 20,000 cells/cm2, appeared to be highly detrimental to the attachment and growth of serially passaged ES colonies of the H9 line (WiCell Research Institute Inc., Wilmington, MA, USA). At the edge of ES colonies that have attached to the higher density feeder layer (32,000 cells/cm2), the ES cells appear to stack up to form a "bulge." This was not observed under the recommended feeder cell density of 20,000 cells/cm2. By contrast, other established ES cell lines are routinely propagated at much higher feeder densities of 60,000 to 70,000 cells/cm2. This report briefly discusses the issue of MEF feeder cell density in relation to our preliminary observations, and the results of other studies.

Animals↗
PubMedDate not supplied

Human embryonic stem cell (hES) colonies display a higher degree of spontaneous differentiation when passaged at lower densities.

Human embryonic stem (hES) cells require cooperative interactions with each other for their survival. Previously, the size of hES cell clumps has been reported to be an important factor in determining their viability during routine serial passage. However, the effects of seeding density of the hES cell clumps per se have not yet been investigated. Therefore, this study attempted to compare the level of spontaneous differentiation of hES colonies passaged at two different split ratios (1:3 and 1:8) of a single confluent well of a six-well dish. After 7 d of in vitro culture following serial passage, hES colonies were assigned into three grades according to their degree of spontaneous differentiation: (1) Grade A, which was completely or mostly undifferentiated; (2) grade B, which was partially differentiated; and (3) grade C, which was mostly differentiated. Assessment of the degree of spontaneous differentiation was based on morphological observations under bright-field and phase-contrast microscopy, as well as on immunocytochemical staining for the pluripotency markers SSEA-3 and TRA-1-81. We observed that, at a split ratio of 1:3, the percentages of grade A, B, and C colonies were 89.5, 8.8, and 1.7%, respectively. This was significantly different from the corresponding values of 52.7, 31.3, and 16.0%, respectively, obtained at a split ratio of 1:8. Hence, our results indicated that a lower passage density led to a higher degree of spontaneous differentiation of hES colonies.

Cell Culture Techniques↗